T Cell Expansion

ABSTRACT

A method for generating or expanding a population of T cells specific for a virus by a method comprising: stimulating T cells by culture in the presence of antigen presenting cells (APCs) presenting a peptide of the virus, wherein at least 10% of the media in which the cells are cultured is conditioned media obtained from a stimulation culture comprising T cells and APCs presenting a peptide of the virus. Also disclosed are methods for accelerating the rate of expansion of a virus-specific T cell population, and methods for treating or preventing diseases or disorders using the generated or expanded T cell population.

FIELD OF THE INVENTION

The present invention relates to methods for generating and/or expandingpopulations of virus-specific T cells.

BACKGROUND TO THE INVENTION

Adoptive transfer of virus-specific T cells is a promising strategy forthe prevention and treatment of viral disease.

Adoptive transfer requires the use of a large number of T cells. Forexample, Chia et al., Mol Ther (2014) 22(1): 132-139 describe treatmentof EBV-positive nasopharyngeal carcinoma (NPC) by adoptive transfer of amedian total number of 9.6×10⁸ EBV-CTLs (ranging from 6.3 to 10.3×10⁸CTLs).

A major problem for therapy by adoptive T cell transfer is the period oftime taken to generate sufficiently large numbers of virus-specific Tcells for administration. Chia et al. (supra) reported that the mediantime taken to produce and the first dose of CTLs was 13 weeks (rangingfrom 8 to 22 weeks). Moreover, it is reported that in this study thatfor 3 patients there was deviation from the scheduled therapy due todelays in CTL production.

SUMMARY OF THE INVENTION

Methods for generating and/or expanding populations of virus-specific Tcells typically include several rounds of stimulation of T cells withantigen presenting cells presenting peptide of the virus of interest(i.e. the virus for which the T cells are specific).

The present invention is based on the finding that populations ofvirus-specific T cells can be expanded more rapidly by performingre-stimulations in the presence of conditioned media obtained from astimulation step. Advantageously, large numbers of virus-specific Tcells can thereby be obtained in a shorter period of time as compared toprior art methods. The present invention is therefore useful forgenerating/expanding populations of virus-specific T cells for use inresearch and/or therapeutic applications.

In one aspect, the present invention provides a method for generating orexpanding a population of T cells specific for a virus, comprisingstimulating T cells by culture in the presence of antigen presentingcells (APCs) presenting a peptide of the virus, wherein at least 10% ofthe media in which the cells are cultured is conditioned media obtainedfrom a stimulation culture comprising T cells and APCs presenting apeptide of the virus.

In another aspect, the present invention provides a method foraccelerating the rate of expansion of a virus-specific T cellpopulation, the method comprising stimulating T cells by culture in thepresence of antigen presenting cells (APCs) presenting a peptide of thevirus, wherein at least 10% of the media in which the cells are culturedis conditioned media obtained from a stimulation culture comprising Tcells and APCs presenting a peptide of the virus.

In another aspect, the present invention provides a method forgenerating or expanding a population of T cells specific for a virus,comprising:

-   -   (i) stimulating T cells by culture in the presence of antigen        presenting cells (APCs) presenting a peptide of the virus; and    -   (ii) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus, wherein at least 10% of        the media in which the cells are cultured is conditioned media        obtained from a stimulation culture of T cells and APCs        presenting a peptide of the virus.

In another aspect, the present invention provides a method forgenerating or expanding a population of T cells specific for a virus,wherein the method comprises:

-   -   (i) stimulating T cells by culture in the presence of antigen        presenting cells (APCs) presenting a peptide of the virus;    -   (ii) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus; and    -   (iii) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus, wherein at least 10% of        the media in which the cells are cultured is conditioned media        obtained from a stimulation culture of T cells and APCs        presenting a peptide of the virus. In some embodiments of the        method the conditioned media is obtained from the stimulation        culture of step (ii).

In another aspect, the present invention provides a method forgenerating or expanding a population of T cells specific for a virus,comprising:

-   -   (i) stimulating T cells by culture in the presence of antigen        presenting cells (APCs) presenting a peptide of the virus;    -   (ii) collecting the cells obtained by step (i), and;    -   (iii) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus, wherein at least 10% of        the media in which the cells are cultured is conditioned media        obtained from a stimulation culture of T cells and APCs        presenting a peptide of the virus.

In another aspect, the present invention provides a method forgenerating or expanding a population of T cells specific for a virus,wherein the method comprises:

-   -   (i) stimulating T cells by culture in the presence of antigen        presenting cells (APCs) presenting a peptide of the virus;    -   (ii) collecting the cells obtained by step (i);    -   (iii) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus;    -   (iv) collecting the cells obtained by step (iii); and    -   (v) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus, wherein at least 10% of        the media in which the cells are cultured is conditioned media        obtained from a stimulation culture of T cells and APCs        presenting a peptide of the virus. In some embodiments of the        method the conditioned media is obtained from the stimulation        culture of step (iii).

In connection with the various aspects of the present invention, in someembodiments the conditioned media is obtained from a stimulation cultureof T cells and APCs presenting a peptide of the virus after a cultureperiod of 1 to 8 days. In some embodiments, the conditioned media isobtained from a stimulation culture of T cells and APCs at aresponder:stimulator ratio of 1:1 to 10:1. In some embodiments, the APCspresenting a peptide of the virus are EBV-transformed lymphoblastoidcell line (LCL) cells. In some embodiments, the at least 10% ofconditioned media is 20 to 40% of conditioned media. In someembodiments, the at least 10% of conditioned media is 10 to 25%,preferably about 15% of conditioned media.

In another aspect, the present invention provides a method forgenerating or expanding a population of Epstein-Barr Virus(EBV)-specific T cells, comprising stimulating T cells by culture in thepresence of EBV-transformed LCLs at a responder to stimulator ratio of2:1 to 7:1 for a period of 1 to 8 days, in media comprising:

-   -   (a) cell culture media comprising 40-50% RPMI-1640 medium,        40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine,    -   (b) at least 10% conditioned media obtained by a method        comprising: stimulating T cells by culture in the presence of        EBV-transformed LCLs at a responder to stimulator ratio of 2:1        to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium,        40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and        added IL-2 at a final concentration of 10-200 IU/ml, for a        period of 1 to 8 days, and    -   (c) added IL-2 at a final concentration of 10-200 IU/ml.

In another aspect, the present invention provides a method foraccelerating the rate of expansion of a population of Epstein-Barr Virus(EBV)-specific T cells, comprising stimulating T cells by culture in thepresence of EBV-transformed LCLs at a responder to stimulator ratio of2:1 to 7:1 for a period of 1 to 8 days, in media comprising:

-   -   (a) cell culture media comprising 40-50% RPMI-1640 medium,        40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine,    -   (b) at least 10% conditioned media obtained by a method        comprising: stimulating T cells by culture in the presence of        EBV-transformed LCLs at a responder to stimulator ratio of 2:1        to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium,        40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and        added IL-2 at a final concentration of 10-200 IU/ml, for a        period of 1 to 8 days, and    -   (c) added IL-2 at a final concentration of 10-200 IU/ml.

In another aspect, the present invention provides a method forgenerating or expanding a population of Epstein-Barr Virus(EBV)-specific T cells, comprising:

-   -   (i) stimulating T cells by culturing PBMCs in the presence of        EBV-transformed LCLs at a responder to stimulator ratio of 10:1        to 80:1 in cell culture media comprising 40-50% RPMI-1640        medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine        for a period of 7 to 14 days;    -   (ii) collecting the cells obtained by step (i);    -   (iii) re-stimulating the T cells by culturing cells collected at        step (ii) in the presence of EBV-transformed LCLs at a responder        to stimulator ratio of 2:1 to 7:1 in cell culture media        comprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20%        FBS, and 1-5 mM L-glutamine, and added IL-2 at a final        concentration of 10-200 IU/ml, for a period of 1 to 8 days;    -   (iv) collecting the cells obtained by step (iii), and;    -   (v) re-stimulating the T cells by culturing cells collected at        step (iv) in the presence of EBV-transformed at a responder to        stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days in        media comprising: (a) cell culture media comprising 40-50%        RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM        L-glutamine, (b) at least 10% conditioned media obtained at the        end point of step (iii), and (c) added IL-2 at a final        concentration of 10-200 (e.g. 40-100) IU/ml. In some        embodiments, the method additionally comprises:    -   (vi) collecting the cells obtained by step (v), and;    -   (vii) re-stimulating the T cells by culturing cells collected at        step (vi) in the presence of EBV-transformed LCLs at a responder        to stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days in        media comprising: (a) cell culture media comprising 40-50%        RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM        L-glutamine, (b) at least 10% conditioned media obtained at the        end point of step (v), and (c) added IL-2 at a final        concentration of 10-200 (e.g. 40-100) IU/ml. In some        embodiments, the method comprises additional steps of collecting        cells, and re-stimulating the T cells by culturing the collected        cells in the presence of EBV-transformed LCLs (e.g. irradiated,        EBV-transformed LCLs) at a responder to stimulator ratio of 2:1        to 7:1 for a period of 1 to 8 days in media comprising: (a) cell        culture media comprising 40-50% RPMI-1640 medium, 40-50% Click's        medium, 5-20% FBS, and 1-5 mM L-glutamine, (b) at least 10%        conditioned media obtained at the end point of the preceding        stimulation step, and (c) added IL-2 at a final concentration of        10-200 IU/ml.

In another aspect, the present invention provides a method of treating acancer in a subject, the method comprising:

-   -   (1) isolating T cells from a subject;    -   (2) generating or expanding a population of T cells specific for        a virus by a method comprising: stimulating T cells by culture        in the presence of antigen presenting cells (APCs) presenting a        peptide of the virus, wherein 10 to 25% of the media in which        the cells are cultured is conditioned media obtained from a        stimulation culture comprising T cells and APCs presenting a        peptide of the virus; and    -   (3) administering the generated or expanded population of T        cells to a subject.

In some embodiments, the conditioned media is obtained from astimulation culture comprising T cells and APCs presenting a peptide ofthe virus at a responder:stimulator ratio of 2:1 to 7:1 for a period of1 to 8 days. In some embodiments, stimulating T cells by culture in thepresence of APCs presenting a peptide of the virus comprises culture inthe presence of EBV-transformed LCLs at a responder to stimulator ratioof 2:1 to 7:1 for a period of 1 to 8 days, in media comprising: (a) cellculture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium,5-20% FBS, and 1-5 mM L-glutamine, (b) 10% to 25% conditioned mediaobtained by a method comprising: stimulating T cells by culture in thepresence of EBV-transformed LCLs at a responder to stimulator ratio of2:1 to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium,40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2at a final concentration of 10-200 IU/ml, for a period of 1 to 8 days,and (c) added IL-2 at a final concentration of 10-200 IU/ml. In someembodiments, the APCs presenting a peptide of the virus areEBV-transformed lymphoblastoid cell line (LCL) cells. In someembodiments, the cancer is an EBV-positive cancer. In some embodiments,the cancer is EBV-positive nasopharyngeal carcinoma (NPC). In someembodiments, about 15% of the media in which the cells are cultured isconditioned media. In some embodiments, step (2) additionally comprises:collecting the generated or expanded population of T cells.

In another aspect, the present invention provides a method of treating acancer in a subject, the method comprising:

-   -   (1) isolating T cells from a subject;    -   (2) generating or expanding a population of T cells specific for        a virus by a method comprising: stimulating T cells by culture        in the presence of antigen presenting cells (APCs) presenting a        peptide of the virus, wherein 10 to 25% of the media in which        the cells are cultured is conditioned media, wherein the        conditioned media is obtained from a stimulation culture        comprising T cells and APCs presenting a peptide of the virus at        a responder:stimulator ratio of 2:1 to 7:1 for a period of 1 to        8 days; and    -   (3) administering the generated or expanded population of T        cells to a subject.

In some embodiments, stimulating T cells by culture in the presence ofAPCs presenting a peptide of the virus comprises culture in the presenceof EBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1for a period of 1 to 8 days, in media comprising: (a) cell culture mediacomprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS,and 1-5 mM L-glutamine, (b) 10% to 25% conditioned media obtained by amethod comprising: stimulating T cells by culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 incell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click'smedium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a finalconcentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c)added IL-2 at a final concentration of 10-200 IU/ml. In someembodiments, the cancer is an EBV-positive cancer. In some embodiments,the cancer is EBV-positive nasopharyngeal carcinoma (NPC). In someembodiments, about 15% of the media in which the cells are cultured isconditioned media. In some embodiments, step (2) additionally comprises:collecting the generated or expanded population of T cells.

In another aspect, the present invention provides a method forgenerating or expanding a population of T cells specific for a virus,comprising stimulating T cells by culture in the presence of antigenpresenting cells (APCs) presenting a peptide of the virus, wherein 10%to 25% of the media in which the cells are cultured is conditioned mediaobtained from a stimulation culture comprising T cells and APCspresenting a peptide of the virus.

In some embodiments, the conditioned media is obtained from astimulation culture comprising T cells and APCs presenting a peptide ofthe virus at a responder:stimulator ratio of 2:1 to 7:1 for a period of1 to 8 days. In some embodiments, stimulating T cells by culture in thepresence of APCs presenting a peptide of the virus comprises culture inthe presence of EBV-transformed LCLs at a responder to stimulator ratioof 2:1 to 7:1 for a period of 1 to 8 days, in media comprising: (a) cellculture media comprising 40-50% RPMI-1640 medium, 40-50% Click's medium,5-20% FBS, and 1-5 mM L-glutamine, (b) 10% to 25% conditioned mediaobtained by a method comprising: stimulating T cells by culture in thepresence of EBV-transformed LCLs at a responder to stimulator ratio of2:1 to 7:1 in cell culture media comprising 40-50% RPMI-1640 medium,40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2at a final concentration of 10-200 IU/ml, for a period of 1 to 8 days,and (c) added IL-2 at a final concentration of 10-200 IU/ml. In someembodiments, the APCs presenting a peptide of the virus areEBV-transformed lymphoblastoid cell line (LCL) cells. In someembodiments, the 10% to 25% of conditioned media is about 15% ofconditioned media. In some embodiments, the method additionallycomprises: collecting the generated or expanded population of T cells.In some embodiments, the method additionally comprises: mixing thegenerated or expanded population of T cells with a pharmaceuticallyacceptable carrier, adjuvant, excipient or diluent.

In another aspect, the present invention provides a population of Tcells specific for a virus, wherein the population of T cells isobtained by, obtainable by, or is the product of, a method according tothe present invention.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a population of T cells according to the presentinvention, and a pharmaceutically acceptable carrier, adjuvant,excipient or diluent.

In another aspect, the present invention provides a population of Tcells or pharmaceutical composition according to the present inventionfor use in the treatment or prevention of a disease or disorder.

In another aspect, the present invention provides the use of apopulation of T cells or pharmaceutical composition according to thepresent invention in the manufacture of a medicament or vaccine for usein the treatment or prevention of a disease or disorder.

In another aspect, the present invention provides a method of treatingor preventing a disease or disorder in a subject, comprisingadministering to a subject a therapeutically or prophylacticallyeffective amount of a population of T cells or pharmaceuticalcomposition according to the present invention.

In some embodiments, the disease or disorder is caused or exacerbated byinfection with the virus for which the T cells are specific, or is adisease or disorder for which infection with the virus for which the Tcells are specific is a risk factor. In some embodiments, the disease ordisorder is a cancer. In some embodiments, the cancer is an EBV-positivecancer. In some embodiments, the cancer is an EBV-positivenasopharyngeal carcinoma (NPC).

In another aspect, the present invention provides a kit of partscomprising a predetermined quantity of a population of T cells orpharmaceutical composition according to the present invention.

DESCRIPTION

T Cells and Antigen Presenting Cells

The present invention is concerned with the generation and/or expansionof populations of virus-specific T cells.

As used herein, a virus-specific T cell is a T cell reactive to cellsinfected with, or comprising a peptide of, a virus. A virus-specific Tcell comprises a T Cell Receptor (TCR) capable of binding to an MHCmolecule presenting a peptide of the virus for which the T cell isspecific.

T Cell Receptors (TCRs) are heterodimeric, antigen-binding moleculestypically comprising an α-chain and a β-chain. In nature, α-chains andβ-chains are expressed at the cell surface of T cells (αβ T cells) as acomplex with invariant CD3 chains. An alternative TCR comprising γ and δchains is expressed on a subset of T cells (γδ T cells). TCRs recognise(bind to) antigen peptide presented by major histocompatibility complex(MHC) molecules. TCR structure and recognition of the peptide-MHCcomplex is described in detail for example in Immunobiology, 5^(th) Edn.Janeway C A Jr, Travers P, Walport M, et al. New York: Garland Science(2001), Chapters 3 and 6, which are hereby incorporated by reference intheir entirety.

T cells may be characterised by reference to surface expression of oneor more of: a TCR polypeptide (e.g. α, β, γ or δ chain), a CD3polypeptide (e.g. γ, δ or ε chain), CD8, and CD4. Surface expression ofa given polypeptide can be measured by various methods well known in theart, e.g. by antibody-based methods such as immunohistochemistry,immunocytochemistry, and flow cytometry.

T cells expanded according to the methods of the present inventioncomprise a TCR specific for a virus. In some embodiments the T cells areCD3+CD8+ T cells. In some embodiments, the T cells are cytotoxic Tcells. In some embodiments, the T cells are CD3+CD4+ T cells. In someembodiments, the T cells are helper T cells. In some embodiments, themethods of the present invention are for generating and/or expanding apopulation of virus-specific cytotoxic T lymphocytes (CTLs) and helper Tlymphocytes.

CTLs are capable of effecting cell death in cells infected with a virusby releasing cytotoxic factors including perforin, granzymes,granulysin, and/or by inducing apoptosis of the infected cell byligating FAS on the infected cell through FASL expressed on the T cell(described for example by Chavez-Galan et al., Cellular and MolecularImmunology (2009) 6(1): 15-25, hereby incorporated by reference in itsentirety). Cytotoxicity can be investigated, for example, using any ofthe methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011),9(6):601-616, hereby incorporated by reference in its entirety. Oneexample of an assay for cytotoxicity of a T cell for to a target cell isthe ⁵¹Cr release assay, in which target cells are treated with ⁵¹Cr,which they internalise. Lysis of the target cells by T cells results inthe release of the radioactive ⁵¹Cr into the cell culture supernatant,which can be detected.

The peptide of the virus presented by the APCs may be derived from aviral particle, or may be encoded by nucleic acid of the virus. As usedherein a “peptide” refers to a chain of two or more amino acid monomerslinked by peptide bonds, which is 50 amino acids or fewer in length.

The viral peptide is presented by an MHC class I or class II molecule.MHC class I molecules are heterodimers of an α-chain and aβ2-microglobulin. The α-chain has three domains designated α1, α2 andα3. The α1 and α2 domains together form the groove to which the peptidepresented by the MHC class I molecule binds, to form the peptide-MHCcomplex. MHC class I α-chains are polymorphic, and different α-chainsare capable of binding and presenting different peptides. Similar to MHCclass I molecules, MHC class II molecules are also heterodimers, andconsist of an α-chain and a β chain. In humans, MHC class I α-chains,and MHC class II α and β-chains are encoded by human leukocyte antigen(HLA) genes.

The virus-specific T cells according to the present disclosure comprisea TCR which is capable of binding to a peptide of the virus for whichthe T cell is specific, presented by an MHC class I or MHC class IImolecule.

The virus for which the T cells are specific may be a dsDNA virus (e.g.adenovirus, herpesvirus, poxvirus), ssRNA virus (e.g. parvovirus), dsRNAvirus (e.g. reovirus), (+)ssRNA virus (e.g. picornavirus, togavirus),(−)ssRNA virus (e.g. orthomyxovirus, rhabdovirus), ssRNA-RT virus (e.g.retrovirus) or dsDNA-RT virus (e.g. hepadnavirus). The presentdisclosure contemplates viruses of the families adenoviridae,herpesviridae, papillomaviridae, polyomaviridae, poxviridae,hepadnaviridae, parvoviridae, astroviridae, caliciviridae,picornaviridae, coronaviridae, flaviviridae, togaviridae, hepeviridae,retroviridae, orthomyxoviridae, arenaviridae, bunyaviridae, filoviridae,paramyxoviridae, rhabdoviridae and reoviridae.

Viruses associated with a disease or disorder are of particularinterest. Accordingly, the following viruses are contemplated:adenovirus, Herpes simplex type 1 virus, Herpes simplex type 2 virus,Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Humanherpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox,Hepatitis B virus, Parvovirus B19, Human Astrovirus, Norwalk virus,coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acuterespiratory syndrome virus, hepatitis C virus, yellow fever virus,dengue virus, West Nile virus, TBE virus, Rubella virus, Hepatitis Evirus, Human immunodeficiency virus, influenza virus, lassa virus,Crimean-Congo hemorrhagic fever virus, Hantaan virus, ebola virus,Marburg virus, measles virus, mumps virus, parainfluenza virus,respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus,orbivirus, coltivirus, and banna virus.

In some embodiments, the virus is Epstein-Barr virus (EBV). Accordingly,in some embodiments the method is for generating or expanding apopulation of EBV-specific T cells.

In some embodiments, the EBV is strain B95-8, P3HR-1, or a derivativethereof.

In the present invention, the viral peptide is presented by an antigenpresenting cell (APC). APCs process polypeptides by the molecularmachinery to peptides which then become associated with MHC moleculesand presented as peptide-MHC complexes at the cell surface. DifferentTCRs display different ability to bind to, and therefore differentreactivity to, different peptide-MHC complexes.

Viral peptides/polypeptides are processed and presented in complex withMHC molecules. Antigen processing, loading and presentation on MHC isdescribed in detail in, for example, Immunobiology, 5^(th) Edn. JanewayC A Jr, Travers P, Walport M, et al. New York: Garland Science (2001),Chapter 5, hereby incorporated by reference in entirety.

Different kinds of T cells are activated through their TCRs byrecognition of MHC-peptide complexes. CD8+ T cells recognize peptide-MHCclass I complexes, whilst CD4+ T cells recognize peptide-MHC class IIcomplexes. T cell activation requires binding MHC-peptide complex forwhich the TCR of the T cell has high affinity in the context of apositive costimulatory signal from APC. The process of T cell activationis well known to the skilled person and described in detail, forexample, in Immunobiology, 5^(th) Edn. Janeway C A Jr, Travers P,Walport M, et al. New York: Garland Science (2001), Chapter 8, which isincorporated by reference in its entirety.

APCs according to the invention may be professional APCs. ProfessionalAPCs are specialised for presenting antigen to T cells; they areefficient at processing and presenting MHC-peptide complexes at the cellsurface, and express high levels of costimulatory molecules.Professional APCs include dendritic cells (DCs), macrophages, and Bcells. Non-professional APCs are other cells capable of presentingMHC-peptide complexes to T cells, in particular MHC Class I-peptidecomplexes to CD8+ T cells.

In connection with the present invention, the APC may be any cell whichis infected with, or comprising or expressing a peptide of, the virusfor which the T cell comprises a specific TCR. The cell infected with,or comprising or expressing a peptide of, the virus may present apeptide of the virus in the context of an MHC molecule at the cellsurface.

It will be appreciated that reference to “a peptide” herein encompassesplural peptides. For example, APCs presenting a peptide of the virus maypresent plural peptides of the virus.

The APCs may comprise an HLA allele encoding a MHC class I or class IImolecule capable of presenting the peptide of the virus for which a TCRof the T cell is specific. In some embodiments the APCs may be HLAmatched for the viral peptide recognised by a TCR of the T cell.

In some embodiments the APCs may express or comprise a peptide of thevirus as a result of being infected with the virus. In some embodimentsthe APCs may have been modified to comprise or express a peptide of thevirus. For example, in some embodiments the cell may have been modifiedto comprise nucleic acid encoding a peptide of the virus, or may havebeen pulsed with a peptide of the virus.

In some embodiments, viral peptide(s) may be provided to the APCs in alibrary of peptide mixtures, which may be referred to as pepmixes. Insome embodiments, there is pooling of a variety of pepmixes for exposureto APCs. APCs presenting a peptide of the virus may be exposed toperipheral blood T-cells under certain conditions to result instimulation of T-cells specific for the certain viral peptide(s).

In some embodiments, the APCs are B cells or are derived from B cells.In particular embodiments, the APCs may be cells of a lymphoblastoidcell line (LCL).

LCLs can be prepared by viral transformation of B cells. LCLs aretypically produced by transformation of B cells with Epstein-Barr virus.Generation and characteristics of LCLs is described in detail, forexample, in Hui-Yuen et al., J Vis Exp (2011) 57: 3321, and Hussain andMulherkar, Int J Mol Cell Med (2012) 1(2): 75-87, both herebyincorporated by reference in entirety. Briefly, LCLs can be produced byincubation of PBMCs with concentrated cell culture supernatant of cellsproducing EBV, for example B95-8 cells, in the presence of cyclosporinA.

In some embodiments the APCs are obtained from, or are derived fromcells obtained from, the same subject as the subject from which thepopulation of T cells is generated or expanded. That is, in someembodiments the APCs and T cells are of autologous origin. In someembodiments, the APCs are obtained from, or are derived from cellsobtained from, a different subject to the subject from which thepopulation of T cells is generated or expanded. That is, in someembodiments the APCs and T cells are of heterologous origin.

In some embodiments according to the present invention, the APCs areLCLs prepared by transformation of B cells with EBV, wherein the B cellsare obtained from the same subject as the T cells used in the methods ofthe invention. In some embodiments the LCLs are prepared bytransformation of B cells obtained from a different subject to thesubject from which the T cells are obtained.

In some embodiments, the B cells used for the preparation of LCLs may beobtained from a population of peripheral blood mononuclear cells (PBMCs)which is obtained from the same individual as the population of PBMCsfrom which the population of virus-specific T cells according to theinvention is generated and/or expanded. In some embodiments, the B cellsused for the preparation of LCLs is obtained from the same population ofPBMCs from which the population of virus-specific T cells according tothe invention is generated and/or expanded.

In some embodiments, the APCs (e.g. the LCLs) are irradiated or treatedwith a substance (e.g. mitomycin C) to prevent their proliferation,prior to culture in the presence of the T cells. Irradiation of LCLs inaccordance with the present methods is typically at 6000 to 12000 rads.

The present methods are useful for producing large numbers ofvirus-specific T cells in a shorter period time relative to prior artmethods for generation/expansion of virus-specific T cells. Inparticular, the methods are useful for producing large numbers ofvirus-specific T cells for adoptive transfer of the virus-specific Tcells for treatment or prophylaxis of diseases caused or exacerbated bythe virus, or for which infection with the virus is a risk factor.

For example, when the methods are for generating and/or expanding apopulation of EBV-specific T cells, the cells are useful to treat an EBVassociated disorder such as nasopharangeal carcinoma (NPC) by adoptivetransfer, e.g. as described in Chia W K et al., Molecular Therapy(2014), 22(1): 132-139, herein incorporated by reference in itsentirety.

Adoptive transfer of T cells generally refers to a process by which Tcells are obtained from a subject, typically by drawing a blood sample.The T cells are then typically treated or altered in some way, andeither returned to the same subject or introduced into a differentsubject. The treatment is typically aimed at providing a T cellpopulation with certain desired characteristics to a subject, orincreasing the frequency of T cells with such characteristics in thatsubject. Adoptive transfer of virus specific T cells is described, forexample, in Cobbold et al., (2005) J. Exp. Med. 202: 379-386 and Rooneyet al., (1998), Blood 92:1549-1555, hereby incorporated by reference inits entirety.

A subject herein may be a human. In some embodiments, a subject may be anon-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent(including any animal in the order Rodentia), cat, dog, pig, sheep,goat, cattle (including cows, e.g. dairy cows, or any animal in theorder Bos), horse (including any animal in the order Equidae), donkey,and non-human primate).

Stimulating T Cells

In the present invention, populations of T cells are generated and/orexpanded. The methods generally comprise steps of stimulating T cells,resulting in their cell division and therefore an increase their number.

A population of T cells may be generated from a single T cell bystimulation and consequent cell division. An existing population of Tcells may be expanded by stimulation and consequent cell division ofcells of the population of T cells.

APCs presenting a peptide of the virus preferentially stimulate T cellsspecific for the virus (i.e. T cells having a TCR capable of recognisingviral peptide presented by the APC), and stimulation therefore causescell division and proliferation of these cells over e.g. other T cellswhich do not comprise/express a TCR specific for the virus. Thepopulation of cells at the end of a stimulation is therefore enrichedfor T cells specific for the virus as compared to the population ofcells prior to stimulation; that is, the virus-specific T cells arepresent at an increased frequency in the population of cells followingstimulation. In this way, a population of T cells specific for the virusis expanded/generated out of a heterogeneous population of T cellshaving different specificities.

T cells are stimulated to proliferate by cell division followingactivation. As described hereinabove, T cell activation occurs throughbinding MHC-peptide complex for which the TCR of the T cell has highaffinity in the context of a positive costimulatory signal from APC.Activation induces T cells to produce IL-2, which promotes celldivision. T cell activation also upregulates expression of the receptorfor IL-2, and so proliferation of activated T cells is promoted in anautocrine fashion.

The methods of the present invention involve steps of stimulating andre-stimulating T cells using APCs presenting a peptide of the virus forwhich the T cell is specific. The stimulating APCs are infected with, orcomprise or express a peptide of, the virus for which the T cellcomprises a specific TCR, and present viral peptide in the context of anMHC molecule. Stimulation promotes cell division (i.e. causes the Tcell(s) to proliferate), resulting in generation and/or expansion of apopulation of T cells specific for the virus.

A method step comprising “stimulating” and/or “re-stimulating” T cellsby culture in the presence of APCs presenting a peptide of the virus maybe referred to herein as a “stimulation step”. The culture of T cellsand APCs presenting a peptide of the virus in the context of astimulation step according to the invention may be referred to herein asa “stimulation culture”. As will be clear from the present disclosure, astimulation culture of a stimulation step comprises cells in culture inmedia comprising cell culture media, and in many cases also comprisingconditioned media.

Stimulating T cells according to the methods of the present inventioninvolves culture of T cells in the presence of the APCs; i.e. co-cultureof the T cells and APCs. Co-culture is typically performed in vitro orex vivo.

In the present methods, a population of virus-specific T cells istypically generated from, or expanded out of, a population of PBMCs.Accordingly, in some embodiments of the methods of the present inventionthe T cells stimulated by culture in the presence of APCs are present inthe culture with other PBMCs such as B cells, NK cells, and/ormonocytes. In some embodiments, the population of T cells may begenerated from, or expanded out of, a population of leukocytes, and maytherefore be in culture with leukocytes other than T cells, such as Bcells, NK cells, monocytes, neutrophils, eosinophils, and/or basophils.

The population of cells from which the population of virus-specific Tcells is generated from, or expanded out of, may be referred to hereinas “responders”, and the APCs presenting a peptide of the virus may bereferred to herein as “stimulators”. In some embodiments, thestimulation steps of the present methods provide “responders” and“stimulators” at particular ratios, referred to herein as the “responderto stimulator ratio”. As will be clear from the above, in someembodiments “responders” as used herein refers to a population of PBMCs.In some embodiments “responders” refers to a population of T cells, orleukocytes.

In the initial stimulation step, the “responders” are the population ofcells comprising the T cells from which the population of virus-specificT cells is to be generated and/or expanded. The “responders” of aninitial stimulation may e.g. be a population of T cells, a population oflymphocytes, a population of leukocytes, or a population of PBMCs. Inparticular embodiments, the “responders” of an initial stimulation are apopulation of PBMCs.

In stimulation steps subsequent to the initial stimulation step (i.e.steps of re-stimulating the T cells), the “responders” are the cells atthe end of the preceding stimulation step, for example the cellscollected at the end of the preceding stimulation step. The “responders”of a subsequent stimulation step will include viable cells in culture atthe end of the preceding stimulation step, including the cells whichhave been stimulated to divide.

For example, in a method comprising the following steps:

-   -   (i) stimulating T cells by culture in the presence of APCs        presenting a peptide of the virus;    -   (ii) collecting the cells obtained by step (i), and;    -   (iii) re-stimulating the T cells by culture in the presence of        APCs presenting a peptide of the virus, wherein at least 10% of        the media in which the cells are cultured is conditioned media        obtained from a stimulation culture of T cells and APCs        presenting a peptide of the virus;

the “responders” for stimulation of step (iii) are the cells collectedat step (ii).

It will be appreciated that the “responder to stimulator ratio” refersto the relative number of cells at the beginning of a given stimulationstep, i.e. the ratio of cells provided to the culture at the start ofthe stimulation step.

In some embodiments the “responders” of a re-stimulation step mayinclude cells other than T cells; e.g. other PBMCs or leukocytes asdescribed above. For example, in embodiments wherein the responders ofan initial stimulation step of a method according to the invention arePBMCs, the cells collected at the end of said initial stimulation stepmay include PBMCs or leukocytes other than T cells.

Methods for expanding virus-specific T cells comprising multiplestimulation steps are well known to the skilled person. Typical cultureconditions (i.e. cell culture media, additives, temperature, gaseousatmosphere), ratios of responders to stimulators, culture periods forstimulation steps, etc. can be readily determined by reference e.g. toBollard et al., J Exp Med (2004), 200(12): 1623-1633 and Straathof etal., Blood (2005), 105(5): 1898-1904, both hereby incorporated byreference in entirety.

Co-culture of T cells and APCs in stimulation steps according to theinvention is performed in cell culture media. The cell culture media canbe any media in which T cells and APCs according to the invention can bemaintained in culture in vitro/ex vivo. Culture media suitable for usein the culture of lymphocytes is well known to the skilled person, andincludes, for example, AIM-V medium, Iscoves medium and RPMI-1640medium.

As used herein, “cell culture media” is distinct from “conditionedmedia”. As will be clear from the fullness of the present disclosure, aculture comprising T cells and APCs according to the present inventionmay be established using substantially only cell culture media (e.g. inan initial stimulation step), or may comprise both cell culture mediaand conditioned media (e.g. in a subsequent stimulation step).

In some embodiments, cell culture media may comprise RPMI-1640 mediumand/or Click's medium (also known as Eagle's Ham's amino acids (EHAA)medium). The compositions of these media are well known to the skilledperson. The formulation of RPMI-1640 medium is described in e.g. Mooreet al., JAMA (1967) 199:519-524, and the formulation of Click's media isdescribed in Click et al., Cell Immunol (1972) 3:264-276. RPMI-1640medium can be obtained from e.g. ThermoFisher Scientific, and Click'smedium can be obtained from e.g. Sigma-Aldrich (Catalog No. C5572).

In some embodiments, the cell culture media comprises RPMI-1640 mediumand Click's medium. In some embodiments the cell culture media comprises(by volume) 25-65% RPMI-1640 medium, and 25-65% Click's medium. In someembodiments the cell culture media comprises 30-60% RPMI-1640 medium,and 30-60% Click's medium. In some embodiments the cell culture mediacomprises 35-55% RPMI-1640 medium, and 35-55% Click's medium. In someembodiments the cell culture media comprises 40-50% RPMI-1640 medium,and 40-50% Click's medium. In some embodiments the cell culture mediacomprises 45% RPMI-1640 medium, and 45% Click's medium.

In some embodiments the cell culture media may comprise one or more cellculture media additives. Cell culture media additives are well known tothe skilled person, and include antibiotics (e.g. penicillin,streptomycin), serum (e.g. fetal bovine serum (FBS), bovine serumalbumin (BSA)), L-glutamine, cytokines/growth factors, etc.

In some embodiments, the cell culture media comprises (by volume) 5-20%FBS, 7.5-15% FBS, or 10% FBS. In some embodiments, the cell culturemedia comprises 1-5 mM L-glutamine, 1.5-3 mM L-glutamine or 2 mMglutamine.

In some embodiments, the cell culture media for a stimulation step maycomprise IL-2. In some embodiments IL-2 may be added (referred to hereinas “added IL-2”). In some embodiments the IL-2 may be exogenous, i.e.IL-2 which is not produced by cells of the culture. In some embodimentsthe IL-2 may be recombinant IL-2.

In some embodiments, the cell culture media of a stimulation stepaccording to the present invention comprises added IL-2 at a finalconcentration of 10-200 IU/ml, 15-175 IU/ml, 20-150 IU/ml, 30-125 IU/ml,or 40-100 UI/ml. The final concentration is the concentration in thetotal volume of media in the culture of the stimulation step (includingthe cell culture media, and any conditioned media).

In some particular embodiments the cell culture media comprises 40-50%RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, 1-5 mM L-glutamine,and added IL-2 at a final concentration of 10-200 UI/ml. In someembodiments the cell culture media comprises 42.5-47.5% RPMI-1640medium, 42.5-47.5% Click's medium, 7.5-15% FBS, 1.5-3 mM L-glutamine andadded IL-2 at a final concentration of 20-150 UI/ml. In some embodimentsthe cell culture media comprises 45% RPMI-1640 medium, 45% Click'smedium, 10% FBS, 2 mM L-glutamine and added IL-2 at a finalconcentration of 40-100 UI/ml.

A stimulation step according to the present invention typically involvesco-culture of the T cells and APCs for a defined period of time.Suitably, the period of time is at least long enough for the APCs tostimulate the T cells to undergo cell division. In some embodiments theperiod of time is long enough for stimulation and at least a single celldivision of a stimulated T cell.

In some embodiments, a stimulation step according to the methods of thepresent invention involves culture of the T cells and APCs for a periodof one of at least 1 hour, at least 6 hours, at least 12 hours, at least24 hours, at least 48 hours, at least 36 hours, at least 72 hours, atleast 4 days, at least 5 days, at least 6 days, at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, or at least 14 days. In some embodiments, a stimulationstep involves culture of the T cells and APCs for a period of one of notmore than 20 days, not more than 14 days, not more than 13 days, notmore than 12 days, not more than 11 days, not more than 10 days, notmore than 9 days, not more than 8 days, not more than 7 days, not morethan 6 days, not more than 5 days, not more than 4 days, or not morethan 72 hours.

In some embodiments, a stimulation step according to the methods of thepresent invention involves culture of the T cells and APCs for a periodof one of 24 hours to 20 days, 48 hours to 14 days, and 3 to 12 days. Insome embodiments, a stimulation step involves culture of the T cells andAPCs for a period of one 7 to 14 days, 8 days to 13 days, and 9 to 12days. In some embodiments, a stimulation step involves culture of the Tcells and APCs for a period of one 1 to 8 days, 2 days to 6 days, and 3to 4 days.

A stimulation step is typically ended by separating the cells in culturefrom the media in which they have been cultured. In some embodiments,the methods comprise a step of collecting the cells at the end of thestimulation step (i.e. collecting the cells obtained at the end of thepreceding stimulation step). The end of a stimulation step is determinedby the culture period for that step; for example, a method stepcomprising stimulating T cells by culture in the presence of APCspresenting a peptide of a virus for a period of 7 days is ended bycollecting the cells at the end of the 7 day culture period.

In some embodiments of the methods according to the present invention astimulation step is ended by diluting the culture, e.g. by the additionof cell culture media. In such embodiments, there need be no collectionof cells, and a re-stimulation step according to the present inventionmay be established by adding cell culture media (and any other additivesas described herein) in an amount appropriate to achieve the desiredpercentages/concentrations of cell culture media, conditioned media (andany additives) for the re-stimulation step.

At the end of the culture period of the stimulation step, the cells maybe collected and separated from the cell culture supernatant. For cellsgrown in suspension culture (such as lymphocytes), the cells may becollected by centrifugation, and the cell culture supernatant may beseparated from the cell pellet. The cell pellet may then be re-suspendedin cell culture media, e.g. for a further stimulation step. In someembodiments, the cells may undergo a washing step after collection. Awashing step may comprise re-suspending the cell pellet in isotonicbuffer such as phosphate-buffered saline (PBS), collecting the cells bycentrifugation, and discarding the supernatant.

Methods for generating and/or expanding a population of T cells specificfor a virus according to the present invention typically involve morethan a single stimulation step. There is no upper limit to the number ofstimulation steps which may be performed in a method according to thepresent invention. In some embodiments the methods comprise more than 2,3, 4 or 5 stimulation steps. In some embodiments, the methods compriseone of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 stimulationsteps.

The stimulation steps in a method according to the present invention maybe different to one another.

The frequency of the virus-specific T cells to be expanded may be verylow in the initial responder population (e.g. the sample of PBMCsobtained from a subject), and in subsequent steps the frequency ofvirus-specific T cells in the responder population is larger as a resultof preceding stimulation(s). Accordingly, in some embodiments themethods provide a higher responder to stimulator ratio for an initialstimulation step than for subsequent (i.e. re-stimulation) steps.

In some embodiments, the ratio of responders to stimulators for aninitial stimulation step is in the range of one of 10:1 to 80:1, 15:1 to70:1, 20:1 to 65:1, 25:1 to 60:1, 30:1 to 50:1, 35:1 to 45:1, or 40:1.In some embodiments, the ratio of responders to stimulators for are-stimulation step is in the range of one of 1:1 to 10:1, 1.5:1 to 8:1,2:1 to 7:1, 2.5:1 to 6:1, 3:1 to 5:1, 3.5:1 to 4.5:1, or 4:1.

An initial stimulation step (i.e. the first stimulation step of themethod) typically involves culture of the T cells and APCs for a periodof time which is a longer period than the culture period for asubsequent stimulation step. The longer period of time allows for anappreciable increase in the number of virus-specific T cells from theirgenerally low frequency in the responder population of the initialstimulation.

In some embodiments, an initial stimulation step involves culture of theT cells and APCs for a period of one of 7 to 14 days, 8 days to 13 days,and 9 to 12 days. In some embodiments, a subsequent stimulation stepinvolves culture of the T cells and APCs for a period of one of 1 to 8days, 2 days to 5 days, and 3 to 4 days.

In some embodiments, the culture of an initial stimulation step may notinclude added IL-2, whereas the culture of subsequent stimulation stepsmay include added IL-2.

In some embodiments, the methods comprise an initial stimulation stepfollowed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 subsequentstimulation steps. In some embodiments, each of the subsequentstimulation steps involves culture of the T cells and APCs for the sameor similar period of time. In some embodiments, each of the subsequentstimulation steps involves culture of the T cells and APCs at the sameor similar ratio of responders to stimulators. In some embodiments, theculture of each of the subsequent stimulation steps comprises added IL-2at the same or similar final concentration.

Conveniently, cultures of cells according to the present disclosure aremaintained at 37° C. in a humidified atmosphere containing 5% CO₂.Cultures can be performed in any vessel suitable for the volume of theculture, e.g. in wells of a cell culture plate, cell culture flasks, abioreactor, etc. The cells of cell cultures according to the presentinvention can be established and/or maintained at any suitable density,as can readily be determined by the skilled person. For example,cultures may be established at an initial density of ˜0.5×10⁶ to ˜5×10⁶cells/ml of the culture (e.g. ˜1×10⁶ cells/ml).

Cells may be cultured in any suitable cell culture vessel. In someembodiments of the methods according to the various aspects of thepresent invention, cells are cultured in a bioreactor. In someembodiments, cells are cultured in a bioreactor described in Somervilleand Dudley, Oncoimmunology (2012) 1(8):1435-1437, which is herebyincorporated by reference in its entirety. In some embodiments cells arecultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100bioreactor.

Conditioned Media

The present invention involves the culture of cells in conditionedmedia. “Conditioned media” refers to media which has been obtained byculture of cells in cell culture media. Conditioned media containsfactors (e.g. cytokines, chemokines, growth factors etc.)secreted/released from the cultured cells. Conditioned media is producedby culturing cells in a culture medium for a time sufficient tocondition the medium, and then collecting the conditioned medium.

Cultures of stimulation steps according to the invention may comprise amixture of a cell culture media and conditioned media. That is, themedia in which the cells of a stimulation culture (e.g. of a stimulationstep) are established and cultured may comprise both cell culture mediaand conditioned media. The use of a mixture of a conditioned media and afresh media may provide a more complex nutrient mixture which isbeneficial to the cells in culture.

It will be appreciated that the composition of the conditioned mediawill depend on the cells, the period of time, the culture conditions,the use of any additives and the composition of the cell culture mediaused for culture from which the conditioned media is obtained.

In the present invention, the conditioned media is obtained from astimulation culture comprising T cells and APCs.

In some embodiments the cell culture media and conditioned media of astimulation culture according to the present methods may be the sameexcept for the conditioned media having been conditioned.

In some embodiments according to the methods of the present invention,the conditioned media may be obtained from the culture of a stimulationstep according to any embodiment described herein.

In some embodiments, the conditioned media is collected at the end pointof a stimulation step. Conveniently, the conditioned media may becollected at the end point of a stimulation step at the time ofcollecting the cells. For example, conditioned media according to theinvention may be obtained from the supernatant obtained bycentrifugation to collect cells at the end of a stimulation step asdescribed herein.

In some embodiments, the conditioned media included in the culture of astimulation step (i.e. a stimulation culture) according to the inventionis obtained from the culture of a stimulation step as described herein.In some embodiments, the conditioned media is not obtained from theculture of an initial stimulation step (i.e. the first stimulation stepof a method according to the invention).

An appropriate culture period to condition a medium may be determined bythe skilled person, based on known methods. Typically, a medium will beconditioned for between about 1 hour and about 8 days, such as betweenabout 1 day to 8 days, 2 days to 5 days, or 3 to 4 days.

In particular embodiments, conditioned media according to the presentinvention is obtained from:

-   -   (1) A stimulation culture of virus-specific T cells in the        presence of APCs presenting a peptide of the virus for which the        T cells are specific;    -   (2) A stimulation culture according to (1), after a culture        period of one of 1 to 8 days, 2 days to 6 days, and 3 to 4 days;    -   (3) A stimulation culture according to (1) or (2), at a        responder:stimulator ratio of one of 1:1 to 10:1, 1.5:1 to 8:1,        2:1 to 7:1, 2.5:1 to 6:1, 3:1 to 5:1, 3.5:1 to 4.5:1, or 4:1;    -   (4) A stimulation culture according to any one of (1) to (3),        wherein the cell culture medium used for the stimulation culture        comprises 30-60% RPMI-1640 medium and/or 30-60% Click's medium        and/or 5-20% FBS and/or 1-5 mM L-glutamine;    -   (5) A stimulation culture according to any one of (1) to (4),        comprising added IL-2 at a final concentration of 10-200 IU/ml,        15-175 IU/ml, 20-150 IU/ml, 30-125 IU/ml, or 40-100 UI/ml;    -   (6) A stimulation culture according to any one of (1) to (5),        additionally comprising at least 10% conditioned media obtained        from a stimulation culture according to any one of (1) to (5);    -   (7) A stimulation culture according to any one of (1) to (6),        wherein the APCs are LCLs, e.g. EBV-transformed LCLs.

In some embodiments, the conditioned media included in the culture of agiven stimulation step may be obtained from the culture of another,different stimulation step of the same method. For example, conditionedmedia included in the culture of a third stimulation step of a methodaccording to the invention may be obtained from the culture of thesecond stimulation step.

In some embodiments, the conditioned media included in the culture of astimulation step may be obtained from the culture of the precedingstimulation step of the method. For example, conditioned media includedin the culture of a third stimulation step may be obtained from theculture of the second stimulation step, and conditioned media includedin the culture of a fourth stimulation step may be obtained from theculture of the third stimulation step, and so on.

In embodiments of the methods according to the present invention, notevery stimulation step comprises culture in media including conditionedmedia. For example, in some embodiments the culture of the initialstimulation step according to the methods does not include conditionedmedia.

A culture of a stimulation step comprising conditioned media may containconditioned media in an amount (by volume) of one of at least 2%, atleast 5%, at least 7.5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95% ofthe total volume of media in the culture of the stimulation step. Theremaining volume may be made-up by cell culture media as describedherein, including any additives thereto. In some embodiments, theconditioned media may comprise at least 10% of the total volume of mediain the culture of the stimulation step.

Although conditioned media is rich in growth factors and cytokines ittypically comprises a lower concentration of e.g. amino acids andglucose as compared to comparable cell culture media which has not beenconditioned, due e.g. to consumption by the cells cultured in themedium. Conditioned media may also comprise waste products of the cellsused to condition the media. Such factors may have a negative impact oncell growth and division, and so it may therefore be important that not100% of the media in a stimulation culture of a stimulation stepaccording to the invention is conditioned media. In some embodiments, aculture of a stimulation step comprising conditioned media may containconditioned media in an amount (by volume) of one of less than 100%,less than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, lessthan 50%, less than 45%, less than 40%, less than 35%, less than 30%,less than 25%, less than 20%, or less than 15% of the total volume ofmedia in the culture of the stimulation step.

In some embodiments, the conditioned media may comprise up to 30%, up to25%, up to 20% or up to 15% of the total volume of media in the cultureof the stimulation step.

In some embodiments, the conditioned media may comprise one of 5 to 70%,7.5 to 70%, 10 to 70%, 10 to 65% 10 to 60%, 12.5 to 55%, 15 to 50%, 15to 45%, 15 to 40%, 17.5% to 45%, 20-40%, 20-35%, or 20-30% of the totalvolume of media in the culture of the stimulation step. In someembodiments, the conditioned media may comprise 20-30% of the totalvolume of media in the culture of the stimulation step. In someembodiments, the conditioned media may comprise 10 to 25%, preferablyabout 15% of the total volume of media in the culture of the stimulationstep.

In some embodiments, the conditioned media may comprise one of 5 to 30%,5 to 27.5%, 5 to 25%, 5 to 22.5%, 5 to 20%, or 5 to 17.5% of the totalvolume of media in the culture of the stimulation step. In someembodiments, the conditioned media may comprise one of 7.5 to 30%, 10 to30% or 12.5 to 30% of the total volume of media in the culture of thestimulation step.

In some embodiments, the conditioned media may comprise one of 5 to 30%,10 to 25%, 10 to 20%, 12.5 to 17.5% or about 15% of the total volume ofmedia in the culture of the stimulation step.

In some embodiments, the conditioned media may comprise one of about 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% of the total volumeof media in the culture of the stimulation step.

Culture media may be a 1× formulation or a concentrated formulation,e.g. a 2× to 250× concentrated medium formulation. In a 1× formulationeach ingredient in the medium is at the concentration intended for cellculture. In a concentrated formulation one or more of the ingredients ispresent at a higher concentration than intended for cell culture.Concentrated culture media is well known in the art. Culture media canbe concentrated using known methods e.g. salt precipitation or selectivefiltration. A concentrated medium may be diluted for use with water(preferably deionized and distilled) or any appropriate solution, e.g.an aqueous saline solution, an aqueous buffer or culture media.

It will be appreciated that the indicated percentages are made withreference to a 1× formulation of conditioned media. The skilled personunderstands that a culture comprising 10% of a 1× formulation ofconditioned media is equivalent to a culture comprising 1% of a 10×concentrated formulation of conditioned media.

In some embodiments, conditioned media may be collected from the cultureof a stimulation step and stored, e.g. at −80° C. The stored conditionedmedia can then be thawed and used in a method according to theinvention. In some embodiments the conditioned media may be treated toremove cells or debris, e.g. by filtration.

Specific Exemplary Embodiments of the Methods

In accordance with the present invention, several specific exemplaryembodiments of the methods are provided. The following specificembodiments are for purely illustrative purposes, to show how thevarious features described hereinabove may be combined, and are notintended to in any way limit the present invention.

A method for generating or expanding, or a method for accelerating therate of expansion of, a population of EBV-specific T cells (e.g. CTLs),comprising stimulating T cells (e.g. within a population of PBMCs) byculture in the presence of EBV-transformed LCLs (e.g. irradiated,EBV-transformed LCLs), at a responder to stimulator ratio of 2:1 to 7:1(e.g. 4:1) for a period of 1 to 8 (e.g. 3 to 4) days, in mediacomprising:

-   -   (a) cell culture media comprising 40-50% (e.g. 45%) RPMI-1640        medium, 40-50% (e.g. 45%) Click's medium, 5-20% (e.g. 10%) FBS,        and 1-5 mM (e.g. 2 mM) L-glutamine,    -   (b) at least 10% (e.g. 10% to 70%) conditioned media obtained by        a method comprising: stimulating T cells (e.g. within a        population of PBMCs) by culture in the presence of        EBV-transformed LCLs (e.g. e.g. irradiated, EBV-transformed        LCLs), at a responder to stimulator ratio of 2:1 to 7:1 (e.g.        4:1) in cell culture media comprising 40-50% (e.g. 45%)        RPMI-1640 medium, 40-50% (e.g. 45%) Click's medium, 5-20% (e.g.        10%) FBS, and 1-5 mM (e.g. 2 mM) L-glutamine, and added IL-2 at        a final concentration of 10-200 (e.g. 40-100) IU/ml, for a        period of 1 to 8 (e.g. 3 to 4) days, and    -   (c) added IL-2 at a final concentration of 10-200 (e.g. 40-100)        IU/ml.

A method for generating or expanding a population of EBV-specific Tcells (e.g. CTLs), comprising:

-   -   (i) stimulating T cells (e.g. within a population of PBMCs) by        culture in the presence of EBV-transformed LCLs (e.g.        irradiated, EBV-transformed LCLs) at a responder to stimulator        ratio of 10:1 to 80:1 (e.g. 40:1) in cell culture media        comprising 40-50% (e.g. 45%) RPMI-1640 medium, 40-50% (e.g. 45%)        Click's medium, 5-20% (e.g. 10%) FBS, and 1-5 mM (e.g. 2 mM)        L-glutamine for a period of 7 to 14 (e.g. 9 to 12) days;    -   (ii) collecting the cells obtained by step (i);    -   (iii) re-stimulating the T cells by culturing cells collected at        step (ii) in the presence of EBV-transformed LCLs (e.g.        irradiated, EBV-transformed LCLs) at a responder to stimulator        ratio of 2:1 to 7:1 (e.g. 4:1) in cell culture media comprising        40-50% (e.g. 45%) RPMI-1640 medium, 40-50% (e.g. 45%) Click's        medium, 5-20% (e.g. 10%) FBS, and 1-5 mM (e.g. 2 mM)        L-glutamine, and added IL-2 at a final concentration of 10-200        (e.g. 40-100) IU/ml, for a period of 1 to 8 (e.g. 3 to 4) days;    -   (iv) collecting the cells obtained by step (iii), and;    -   (v) re-stimulating the T cells by culturing cells collected at        step (iv) in the presence of EBV-transformed LCLs (e.g.        irradiated, EBV-transformed LCLs) at a responder to stimulator        ratio of 2:1 to 7:1 (e.g. 4:1) for a period of 1 to 8 (e.g. 3        to 4) days in media comprising: (a) cell culture media        comprising 40-50% (e.g. 45%) RPMI-1640 medium, 40-50% (e.g. 45%)        Click's medium, 5-20% (e.g. 10%) FBS, and 1-5 mM (e.g. 2 mM)        L-glutamine, (b) at least 10% (e.g. 10% to 70%) conditioned        media obtained at the end point of step (iii), and (c) added        IL-2 at a final concentration of 10-200 (e.g. 40-100) UI/ml.

In some embodiments, the method additionally comprises:

-   -   (vi) collecting the cells obtained by step (v), and;    -   (vii) re-stimulating the T cells by culturing cells collected at        step (vi) in the presence of EBV-transformed LCLs (e.g.        irradiated, EBV-transformed LCLs) at a responder to stimulator        ratio of 2:1 to 7:1 (e.g. 4:1) for a period of 1 to 8 (e.g. 3        to 4) days in media comprising: (a) cell culture media        comprising 40-50% (e.g. 45%) RPMI-1640 medium, 40-50% (e.g. 45%)        Click's medium, 5-20% (e.g. 10%) FBS, and 1-5 mM (e.g. 2 mM)        L-glutamine, (b) at least 10% (e.g. 10% to 70%) conditioned        media obtained at the end point of step (v), and (c) added IL-2        at a final concentration of 10-200 (e.g. 40-100) UI/ml.

In some embodiments, the method comprises additional steps of collectingcells, and re-stimulating the T cells by culturing the collected cellsin the presence of EBV-transformed LCLs (e.g. irradiated,EBV-transformed LCLs) at a responder to stimulator ratio of 2:1 to 7:1(e.g. 4:1) for a period of 1 to 8 (e.g. 3 to 4) days in mediacomprising: (a) cell culture media comprising 40-50% (e.g. 45%)RPMI-1640 medium, 40-50% (e.g. 45%) Click's medium, 5-20% (e.g. 10%)FBS, and 1-5 mM (e.g. 2 mM) L-glutamine, (b) at least 10% (e.g. 10% to70%) conditioned media obtained at the end point of the precedingstimulation step, and (c) added IL-2 at a final concentration of 10-200(e.g. 40-100) UI/ml.

Rates of Expansion

The present methods achieve an improved rate of expansion forpopulations of virus-specific T cells as compared to prior art methods.

The rate of expansion for a T cell population can be analysed by methodswell known to the skilled person. Methods include measuring the numberof T cells at one or more time points.

For example, the number of T cells can be determined after performing amethod according to the invention and compared to the number of T cellsat the beginning of the method; fold expansion in the number of T cellscan then be calculated.

Rates of expansion can also be determined by analysing cell division byT cells over a period of time. Cell division for a given population of Tcells can be analysed, for example, by in vitro analysis ofincorporation of ³H-thymidine or by CFSE dilution assay, e.g. asdescribed in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564,hereby incorporated by reference in entirety.

The improvement in the rate of expansion achieved by the methodsaccording to the present invention can be determined by performing amethod according to the invention, and comparing the expansion for Tcells in that method to a comparable, control method lacking astimulation culture in the presence of conditioned media.

For example, the rate of expansion can be compared between two methods:(i) a method according to the invention, comprising stimulating T cellsby culture in the presence of APCs presenting a peptide of the virus,wherein at least 10% of the media in which the cells are cultured isconditioned media obtained from a stimulation culture comprising T cellsand APCs presenting a peptide of the virus; and (ii) a comparablecontrol method, which is the same except that cell culture media is usedin place of the conditioned media.

In some embodiments, the rate of expansion for a population of T cellsin a method according to the present invention is one of at least 1.001times, 1.002 times, 1.003 times, 1.004 times, 1.005 times, 1.006 times,1.007 times, 1.008 times, 1.009 times, 1.01 times, 1.02 times, 1.03times, 1.04 times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times,1.7 times, 1.8 times, 1.9 times, or 2 times the rate of expansion in acomparable control method in which cell culture media is used in placeof the conditioned media.

The rate of expansion may be of the virus-specific T cell population, orthe total T cell population.

Properties of the Expanded Cells

Advantageously, the virus-specific T cells generated/expanded accordingto the method of the present invention retain the same functionalproperties as virus-specific T cells generated/expanded according toprior art methods. That is, the accelerated rate of expansion does notnegatively influence the functional properties of the expanded T cells.

For example, in embodiments wherein the methods generate/expand apopulation of virus-specific CTLs, the CTLs display similar cytotoxicityto cells infected with or comprising/expressing a peptide of the virusas virus-specific CTLs expanded according to methods lacking stimulationculture in the presence of conditioned media.

Cytotoxicity of expanded CTLs can be analysed e.g. by culturing theexpanded T cell population with APCs presenting a peptide of the virusfor which the T cell is specific at different effector (i.e. T cell) totarget (i.e. APC) ratios, and measuring specific lysis of the APCs. Forexample, cytotoxicity of an EBV-specific CTL population can be analysedby measuring specific lysis of EBV-transformed LCL cells at differenteffector to target ratios.

Therapeutic and Prophylactic Applications

The present methods find application in methods of medical treatment.Treatment may be provided to subjects having a disease or condition inneed of treatment.

In particular, methods for treating or preventing a disease or disorder(e.g. a cancer) are provided comprising a method for generating orexpanding a population of T cells specific for a virus, or a method foraccelerating the rate of expansion of a virus-specific T cellpopulation, as described herein.

Methods of treatment or preventing a disease or disorder according tothe present invention may comprise adoptive transfer of T cells.

In one aspect, the present invention provides a method of treating orpreventing a disease or disorder in a subject, comprising:

-   -   (1) isolating T cells from a subject;    -   (2) generating or expanding a population of T cells specific for        a virus as described herein, and;    -   (3) administering the generated or expanded population of T        cells to a subject.

In some embodiments, the subject from which the T cells are obtained atstep (1) of the method is the same subject as the subject to which thepopulation of T cells generated or expanded according to the methodsdescribed herein are administered at step (3) of the method (i.e.adoptive transfer is of autologous T cells). In some embodiments, thesubject from which the T cells are obtained at step (1) of the method isa different subject to the subject to which the population of T cellsgenerated or expanded according to the methods described herein areadministered at step (3) of the method (i.e., adoptive transfer is ofallogenic T cells).

It will be appreciated that the T cells isolated from the subjectaccording to step (1) above may be within a population of PBMCs.

In some embodiments, the method may comprise one or more of thefollowing steps: taking a blood sample from a subject; isolating PBMCsfrom the blood sample; isolating T cells from the blood sample;generating or expanding a population of T cells specific for a virusaccording to the methods described herein; modifying the T cells, e.g.to express a chimeric antigen receptor (CAR) or T cell receptor (TCR);collecting the generated/expanded population of virus-specific T cells;mixing the generated/expanded population of virus-specific T cells withan adjuvant, diluent, or carrier; administering the generated/expandedpopulation of virus-specific T cells to a subject.

The methods may comprise modification or treatment of the T cells insome way. For example, the T cell may be modified in vitro or ex vivo toexpress or comprise a chimeric antigen receptor (CAR) or T cell receptor(TCR). T cells can be modified according to methods well known to theskilled person. The modification may comprise nucleic acid transfer forpermanent or transient expression of the transferred nucleic acid. Anysuitable genetic engineering platform may be used for such modification.Suitable methods include the use of genetic engineering platforms suchas gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNAtransfection, transposon-based gene delivery and RNA transfection, forexample as described in Maus et al., Annu Rev Immunol (2014) 32:189-225,hereby incorporated by reference in its entirety.

The treatment may be aimed at prevention of a disease/disorder, and assuch the virus-specific T cells generated/expanded according to themethod of the present invention may be employed prophylactically againstdevelopment of a disease state. This may take place before the onset ofsymptoms of the disease state, and/or may be given to subjectsconsidered to be at greater risk of the disease or disorder.

Through administration of a population of T cells according to theinvention, or administration of a pharmaceutical composition comprisinga population of T cells according to the invention, the disease ordisorder is treated or prevented.

The disease or condition may be one which is caused or exacerbated byinfection with a virus for which the T cells generated/expandedaccording to the method of the present invention are specific. In someembodiments, the disease or condition may be one which is caused orexacerbated by infection with a virus described herein.

In particular, the disease or condition may be one which is caused orexacerbated by Epstein-Barr virus (EBV), influenza virus, measles virus,hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiencyvirus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplexvirus (HSV) or human papilloma virus (HPV).

Virus-specific T cells generated/expanded according to the method of thepresent invention are useful in methods for the treatment or preventionof cancer. Accordingly, in some embodiments the method of treatment orprevention of a disease or disorder is for treating or preventing acancer.

Methods of medical treatment may involve treatment of cancer by a methodof ameliorating, treating, or preventing a malignancy in a human subjectwherein the steps of the method assist or boost the immune system ineradicating cancerous cells. Such methods may include the administrationof virus-specific T cells generated/expanded according to the method ofthe present invention that invoke an active (or achieve a passive)immune response to destroy cancerous cells. Methods of treatment mayoptionally include the co-administration of biological adjuvants (e.g.,interleukins, cytokines, Bacillus Comette-Guerin, monophosphoryl lipidA, etc.) in combination with conventional therapies for treating cancersuch as chemotherapy, radiation, or surgery. Methods of treatment mayinvolve administering virus-specific T cells generated/expandedaccording to the method of the present invention as a vaccine that worksby activating the immune system to prevent or destroy cancer cellgrowth. Methods of medical treatment may involve in vivo, ex vivo, andadoptive immunotherapies, including those using autologous and/orheterologous cells or immortalized cell lines.

A cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervoussystem, peritoneum, pleura, prostate, salivary gland, sigmoid colon,skin, small intestine, soft tissues, spleen, stomach, testis, thymus,thyroid gland, tongue, tonsil, trachea, uterus, vulva, white bloodcells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer, colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancerand sarcoma.

In particular, treatment of head and neck cancer, nasopharyngealcarcinoma (NPC), oropharyngeal cancer (OPC), cervical cancer (CC),gastric/stomach cancer or lung cancer is contemplated.

In some embodiments the cancer is an EBV- or HPV-positive cancer. Insome embodiments, the cancer is EBV-positive NPC. In some embodiments,the cancer is HPV-positive OPC or HPV-positive CC.

Administration of virus-specific T cells generated/expanded according tothe method of the present invention is preferably in a “therapeuticallyor prophylactically effective amount”, this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofthe disease being treated. Prescription of treatment, e.g. decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams & Wilkins.

The present invention provides populations of T cells generated orexpanded according to the methods described herein. Accordingly, the apopulation of T cells specific for a virus, wherein the population of Tcells is obtained by, obtainable by, or is the product of, a methodaccording to the present invention is provided.

Also provided are uses of the populations of T cells in methods for thetreatment or prevention of a disease or disorder as described herein.

That is, the present invention provides a population of T cells specificfor a virus for use in a method of treatment or prevention of a diseaseor disorder, wherein the population of T cells is obtained by,obtainable by, or is the product of, a method according to the presentinvention.

Also provided is a method of treating or preventing a disease ordisorder in a subject, comprising administering a population of T cellsspecific for a virus to a subject, wherein the population of T cells isobtained by, obtainable by, or is the product of, a method according tothe present invention.

Also provided is the use of a population of T cells or pharmaceuticalcomposition according to the present invention in the manufacture of amedicament or vaccine for use in the treatment or prevention of adisease or disorder, wherein the population of T cells is obtained by,obtainable by, or is the product of, a method according to the presentinvention.

Pharmaceutically Useful Compositions and Medicaments

Virus-specific T cells generated/expanded according to the method of thepresent invention may be formulated for clinical use and may comprise apharmaceutically acceptable carrier, diluent, excipient or adjuvant.

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions comprisingvirus-specific T cells generated/expanded according to the method of thepresent invention, such methods of production may comprise one or moresteps selected from: generating/expanding a population of virus-specificT cells according to the methods of the present invention; and/or mixingvirus-specific T cells generated/expanded according to the methods ofthe present invention with a pharmaceutically acceptable carrier,adjuvant, excipient or diluent.

In one aspect, the present invention provides a method of preparing apharmaceutical composition, medicament or vaccine, the method comprisinggenerating or expanding a population of T cells specific for a virusaccording to the methods of the present invention, and mixing the cellsobtained with a pharmaceutically acceptable carrier, adjuvant, diluentor excipient.

The present invention also provides pharmaceutical compositionscomprising a population of T cells according to the present invention,and a pharmaceutically acceptable carrier, adjuvant, excipient ordiluent. The population of T cells may be obtained by, obtainable by, orthe product of, a method according to the present invention.

The present invention provides a pharmaceutical composition according tothe invention for use in a method of treatment or prevention of adisease or disorder.

Also provided is a method of treating or preventing a disease ordisorder in a subject, comprising administering a pharmaceuticalcomposition according to the invention to a subject.

Also provided is the use of a pharmaceutical composition according tothe invention in the manufacture of a medicament or vaccine for use inthe treatment or prevention of a disease or disorder.

Subjects

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient.

A subject may have been diagnosed with a disease or condition requiringtreatment, may be suspected of having such a disease or condition, ormay be at risk of developing such a disease or condition.

Kits

In one aspect of the present invention a kit of parts is provided. Thekit of parts comprises a population of T cells according to the presentinvention, or a pharmaceutical composition according to the presentinvention.

In some embodiments, the kit may include instructions for using thepopulation of T cells or pharmaceutical composition to treat or preventa disease or disorder as described herein.

In some embodiments, a kit may comprise a container comprising aquantity of T cells obtained by a method of the present inventionformulated for administration to a subject (e.g. by admixture with asuitable carrier, excipient, diluent, or adjuvant) preferably byinfusion, more preferably for administration by infusion in a method ofautologous adoptive cellular immunotherapy. The kit may be maintained ata predetermined temperature, e.g. less than about 4° C., less than about−2° C. or less than about −50° C. The kit may further compriseinstructions for the storage and/or transport of the kit and/or for theadministration of the T-cells.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1A and FIG. 1B. Graph showing the effect of conditioned media onEBV-specific T cell expansion from cells obtained from (1A) Donor 1 and(1B) Donors 2 and 3. Increasing percentages of conditioned media areadded to culture media. After one week of co-culture with LCL, thenumber of expanded T cells is compared to the original number of seededT cells.

FIG. 2. Graph showing the effect of conditioned media on EBV-specific Tcell expansion from cells obtained from three donors, in a Grex-100bioreactor. Increasing percentages of conditioned media are added toculture media. After one week of co-culture with LCL, the number ofexpanded T cells is compared to the original number of seeded T cells.

FIG. 3. Graph showing specific killing of EBV-transformed LCL cells byEBV-Specific T cells. T cells expanded by culture in 0% and 30%conditioned medium are co-cultured with LCLs at differenteffector/target cell ratios, and specific lysis of the LCL cells ismeasured after 4 hours.

FIG. 4. Graph showing the effect of the presence of differentpercentages of conditioned media on fold expansion of EBV-specific CTLs,cultured in Grex-100 bioreactor culture vessels.

EXAMPLES

In the following Examples the inventors describe expansion ofEBV-specific T cells including culture in the presence of conditionedmedia. The inventors describe experiments for determining the optimumpercentage of conditioned media to include in cultures, and forconfirming cytotoxic activity of the expanded CTLs.

Example 1: Optimum Percentage of Conditioned Media

To determine the optimum percentage of conditioned medium for T cellexpansion, in T cell expansions performed in 24 well plates, cellculture media comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mML-glutamine is mixed with 10%, 20%, 30%, 40%, 50%, and 60% ofconditioned medium obtained from a co-culture of T cells with LCL cells.

After one week of co-culture with LCL cells, the number of T cells iscounted and compared to the number of seeded cells, to determine foldexpansion.

The expected expansion results are shown on FIG. 1A. T cells areexpanded at a faster rate by the methods including culture in thepresence of conditioned media. The optimum percentage of conditionedmedia in a method step according to B for greatest fold expansion isexpected to be 20-30% conditioned media.

The same experiment is performed on cells obtained from two further,different donors (Donors 2 and 3). It is expected that the results willbe similar as for the Donor 1, with maximum T cell expansion observed at20-30% conditioned media (see FIG. 1B).

Because large-scale expansion of T cells will be performed inbioreactors such as e.g. Grex-100 culture vessels, the optimisedpercentage of conditioned media is further investigated in bioreactorculture.

For Illustrative Purposes:

Cells are cultured at a cell density of 1×10⁶ cells/ml in 200 ml ofmedia, of which varying percentages are conditioned media:

Cell culture media comprising 45% RPMI 1640, 45% Click's Conditioned %Conditioned media, 10% FBS, 2 mM L-glutamine media media 160 ml 40 ml20% 140 ml 60 ml 30% 120 ml 80 ml 40% 100 ml 100 ml  50%  80 ml 120 ml 60%

The expected results for T cell expansion are shown in FIG. 2. It isexpected that the optimum percentage of conditioned media will be20-30%, consistent across different donors.

Example 2: Generation of EBV-Transformed LCLs and Expansion ofEBV-Specific CTLs

Peripheral blood (40-60 mL) obtained from patients with EBV-positive NPCare used to generate both EBV-transformed lymphoblastoid B-cell lines(LCLs) and EBV-specific T cells.

Generation of EBV-Transformed LCLs

Briefly, for LCL generation, 15×10⁶ peripheral blood mononuclear cells(PBMCs) are incubated with concentrated supernatant of B95-8 cultures,in the presence of 1 μg/mL cyclosporin A (Sandoz, Vienna, Austria) toestablish an LCL.

LCLs are irradiated at 60 Gy prior to being used for stimulations (onthe day of the stimulation).

Expansion of EBV-Specific T Cells

Expansion of EBV-specific T cells by two different methods are compared.

In both methods, a first stimulation is performed as follows:

-   -   60×10⁶ PBMCs are re-suspended in cell culture media comprising        45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and        a viable cell count is performed.    -   PBMCs are seeded at 2×10⁶ cells/well into wells of a 24 well        plate.    -   PBMCs are stimulated with irradiated, Acyclovir-treated        autologous LCLs at a responder to stimulator ratio of 40:1.    -   The cells are cultured for 9-12 days at 37° C. in a 5% CO₂        atmosphere.

In both methods, a second stimulation is then performed, as follows:

-   -   Cells are collected at the end of the first stimulation, a        viable cell count is performed, and the cells are re-suspended        at 1×10⁶ cells/ml in cell culture media comprising 45% RPMI        1640, 45% Click's media, 10% FBS, 2 mM L-glutamine.    -   1 ml of cells are added into wells of 24 well plates, or the        cell suspension is added into bioreactors at a concentration of        15×10⁶ cells/GRex10, in cell culture media comprising 45% RPMI        1640, 45% Click's media, 10% FBS, 2 mM L-glutamine, and the        cells are re-stimulated with autologous irradiated LCLs at a        responder to stimulator ratio of 4:1.    -   The cells are cultured for 3-4 days, and then the cells are        re-suspended in fresh cell culture media comprising 45% RPMI        1640, 45% Click's media, 10% FBS, 2 mM L-glutamine. Recombinant        human IL-2 (rhlL-2, Proleukin; Chiron Emeryville, Calif.)) is        added to the cell culture at a final concentration of 40-100        IU/ml.

Subsequent Stimulations

Following the second stimulation, cells are collected, a viable cellcount is performed.

Culture media (i.e. conditioned media) is retained for use in method Bbelow. The cells are then re-suspended at 1×10⁶ cells/ml (in 24 wellplate) or 0.5×10⁶ cells/ml (in GRex10) in cell culture media comprising45% RPMI 1640, 45% Click's media, 10% FBS, 2 mM L-glutamine andre-stimulated with autologous irradiated LCLs at a responder tostimulator ratio of 4:1.

Once 200×10⁶ cells are achieved, cells are then stimulated according tomethod step A or method step B:

Method Step A Method Step B 100 × 10⁶ cells are re-suspended in 200 ml100 × 10⁶ cells are re-suspended in: cell culture media comprising 45%RPMI (1) 180 ml cell culture media comprising 1640, 45% Click's media,10% FBS, 2 mM L- 45% RPMI 1640, 45% Click's media, glutamine,transferred to GRex 100, and re- 10% FBS, 2 mM L-glutamine, and;stimulated with and the cells are re- (2) 20 ml conditioned mediaobtained stimulated with autologous irradiated LCLs at from the cultureat the end of the a responder to stimulator ratio of 4:1, with secondstimulation (i.e. 10% added rhlL-2 at a final concentration of 40-conditioned media), 100 IU/ml. transferred to GRex 100, andre-stimulated The cells are cultured for 3-4 days at 37° C. in with andthe cells are re-stimulated with a 5% CO₂ atmosphere. autologousirradiated LCLs at a responder to stimulator ratio of 4:1, with addedrhlL-2 at a final concentration of 40-100 IU/ml. The cells are culturedfor 3-4 days at 37° C. in a 5% CO₂ atmosphere.

At the end of a stimulation according to method step A or method step B,cells are collected and re-stimulated according to the same method stepA or B.

For re-simulations according to method step B, conditioned media used isfrom the preceding stimulation according to B.

Example 3: Test of Efficacy

The cytotoxic activity of CTLs expanded by methods including culture inthe presence of conditioned media is compared to the cytotoxic activityof CTLs expanded without culture in the presence of conditioned media.

T cells expanded from 0% and 30% conditioned media are added to LCLcells at different Effector/Target cell ratios (E/T ratio), and after 4hours, specific lysis of the LCL cells is measured. It is expected thatthere will be very little or no difference between the specific lysisfor cells expanded by culture in 0% and 30% conditioned media (FIG. 3).

CTLs expanded at an increased rate are expected to retain the ability tospecifically kill EBV-transformed LCL cells.

Example 4: Optimization of Epstein-Barr Virus-Specific T Cells (EB-VSTs)Growth Using Conditioned Medium

The following example describes an investigation of the optimalpercentage of conditioned media to be included in restimulations formaximum CTL expansion.

Week 1:

-   -   Lymphoblastoid cell line (LCL) samples were thawed and cultured        for 1 week    -   Requires minimally 10×10⁶ LCLs to initiate experiment

Week 2:

-   -   Frozen EB-VST03 cells from different patients were thawed,        re-suspended in cell culture media comprising 45% RPMI 1640, 45%        Click's media, 10% FBS, 2 mM L-glutamine, and a viable cell        count was performed        -   EB-VST03 cells were obtained by culture of PMBCs according            to Example 2, wherein the PBMCs underwent first and second            stimulations, and then a third stimulation according to            Method Step A (i.e. in the absence of conditioned media),            after which point cells were harvested and frozen    -   EB-VST03 cells were seeded at 1×10⁶ cells/well of a 24-well        plate    -   Cell suspensions were added to bioreactors (15×10⁶        cells/G-Rex 10) in cell culture media comprising 45% RPMI 1640,        45% Click's media, 10% FBS, 2 mM L-glutamine, and re-stimulated        with autologous irradiated LCLs at a Responder:Stimulator ratio        of 4:1.    -   The cells were cultured for 3-4 days, and then resuspended in        cell culture media comprising 45% RPMI 1640, 45% Click's media,        10% FBS and 2 mM L-glutamine.    -   IL-2 was then added to the cell culture at a final concentration        of 40-100 IU/ml.

Week 3 Onwards:

-   -   Cells were harvested, pooled, counted and stimulated as        described under week 2 above until there were 120×10⁶ cells.    -   100×10⁶ cells were then seeded into G-Rex 100 flasks in cell        culture media comprising 45% RPMI 1640, 45% Click's media, 10%        FBS, 2 mM L-glutamine, and different percentages of conditioned        media (obtained from the culture at the end of the preceding        stimulation culture), as follows:

Cell culture media comprising 45% RPMI 1640, 45% Click's Conditioned %Conditioned media, 10% FBS, 2 mM L-glutamine media media 200 ml  0 ml 0% 170 ml 30 ml 15% 140 ml 60 ml 30% 110 ml 90 ml 45%

-   -   The cells were then cultured for 3-4 days at 37° C. in a 5% CO₂        atmosphere.    -   IL-2 was then added to the cell culture at a final concentration        of 40-100 IU/ml.    -   After one week, the cells were harvested and a viable cell count        was performed.

All viabilities were observed to be greater than 70%. The results areshown in in FIG. 4.

It was found that culture in cell culture media comprising 45% RPMI1640, 45% Click's media, 10% FBS, 2 mM L-glutamine and up to 15%conditioned media gave the highest yield of EB-VSTs, which was ˜20%higher than the rate of EB-VST as compared to using 100% of cell culturemedia comprising 45% RPMI 1640, 45% Click's media, 10% FBS, 2 mML-glutamine.

Fold expansion of EB-VSTs decreased when higher percentages (i.e. 30%,45%) of conditioned media were used.

Fold expansion observed for Sample 3—EB-VST was not as pronounced asobserved for Sample 1 & Sample 2. This might be because Sample 3 EBV-VSTunderwent an additional cycle of stimulation, with the result that cellgrowth was overstressed.

It is concluded that a combination of 15% conditioned media with 85%fresh culture media (complete Click's medium) produced highest EB-VSTyield i.e. higher EB-VST expansion rate as compared to using 100% freshculture media.

Whilst the effect of conditioned medium on EB-VST growth varies amongdifferent individuals, it was observed that in every case, using 15%conditioned medium gave the best expansion rate.

1. A method for generating or expanding a population of T cells specificfor a virus, comprising stimulating T cells by culture in the presenceof antigen presenting cells (APCs) presenting a peptide of the virus,wherein at least 10% of the media in which the cells are cultured isconditioned media obtained from a stimulation culture comprising T cellsand APCs presenting a peptide of the virus.
 2. A method for acceleratingthe rate of expansion of a virus-specific T cell population, the methodcomprising stimulating T cells by culture in the presence of antigenpresenting cells (APCs) presenting a peptide of the virus, wherein atleast 10% of the media in which the cells are cultured is conditionedmedia obtained from a stimulation culture comprising T cells and APCspresenting a peptide of the virus.
 3. A method for generating orexpanding a population of T cells specific for a virus, comprising: (i)stimulating T cells by culture in the presence of antigen presentingcells (APCs) presenting a peptide of the virus; and (ii) re-stimulatingthe T cells by culture in the presence of APCs presenting a peptide ofthe virus, wherein at least 10% of the media in which the cells arecultured is conditioned media obtained from a stimulation culture of Tcells and APCs presenting a peptide of the virus.
 4. A method forgenerating or expanding a population of T cells specific for a virus,comprising: (i) stimulating T cells by culture in the presence ofantigen presenting cells (APCs) presenting a peptide of the virus; (ii)collecting the cells obtained by step (i), and; (iii) re-stimulating theT cells by culture in the presence of APCs presenting a peptide of thevirus, wherein at least 10% of the media in which the cells are culturedis conditioned media obtained from a stimulation culture of T cells andAPCs presenting a peptide of the virus.
 5. A method for generating orexpanding a population of T cells specific for a virus, wherein themethod comprises: (i) stimulating T cells by culture in the presence ofantigen presenting cells (APCs) presenting a peptide of the virus; (ii)collecting the cells obtained by step (i); (iii) re-stimulating the Tcells by culture in the presence of APCs presenting a peptide of thevirus; (iv) collecting the cells obtained by step (iii); and (v)re-stimulating the T cells by culture in the presence of APCs presentinga peptide of the virus, wherein at least 10% of the media in which thecells are cultured is conditioned media obtained from a stimulationculture of T cells and APCs presenting a peptide of the virus.
 6. Themethod according to paragraph 5, wherein the conditioned media isobtained from the stimulation culture of step (iii).
 7. The methodaccording to any one of paragraphs 1 to 6, wherein the conditioned mediais obtained from a stimulation culture of T cells and APCs presenting apeptide of the virus after a culture period of 1 to 8 days.
 8. Themethod according to any one of paragraphs 1 to 7, wherein theconditioned media is obtained from a stimulation culture of T cells andAPCs at a responder:stimulator ratio of 1:1 to 10:1.
 9. The methodaccording to any one of paragraphs 1 to 8, wherein the APCs presenting apeptide of the virus are EBV-transformed lymphoblastoid cell line (LCL)cells.
 10. The method according to any one of paragraphs 1 to 9, whereinthe at least 10% of conditioned media is 20 to 40% of conditioned media.11. A method for generating or expanding a population of Epstein-BarrVirus (EBV)-specific T cells, comprising stimulating T cells by culturein the presence of EBV-transformed LCLs at a responder to stimulatorratio of 2:1 to 7:1 for a period of 1 to 8 days, in media comprising:(a) cell culture media comprising 40-50% RPMI-1640 medium, 40-50%Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, (b) at least 10%conditioned media obtained by a method comprising: stimulating T cellsby culture in the presence of EBV-transformed LCLs at a responder tostimulator ratio of 2:1 to 7:1 in cell culture media comprising 40-50%RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mML-glutamine, and added IL-2 at a final concentration of 10-200 IU/ml,for a period of 1 to 8 days, and (c) added IL-2 at a final concentrationof 10-200 IU/ml.
 12. A method for accelerating the rate of expansion ofa population of Epstein-Barr Virus (EBV)-specific T cells, comprisingstimulating T cells by culture in the presence of EBV-transformed LCLsat a responder to stimulator ratio of 2:1 to 7:1 for a period of 1 to 8days, in media comprising: (a) cell culture media comprising 40-50%RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mML-glutamine, (b) at least 10% conditioned media obtained by a methodcomprising: stimulating T cells by culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 incell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click'smedium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a finalconcentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c)added IL-2 at a final concentration of 10-200 IU/ml.
 13. A method forgenerating or expanding a population of Epstein-Barr Virus(EBV)-specific T cells, comprising: (i) stimulating T cells by culturingPBMCs in the presence of EBV-transformed LCLs at a responder tostimulator ratio of 10:1 to 80:1 in cell culture media comprising 40-50%RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mML-glutamine for a period of 7 to 14 days; (ii) collecting the cellsobtained by step (i); (iii) re-stimulating the T cells by culturingcells collected at step (ii) in the presence of EBV-transformed LCLs ata responder to stimulator ratio of 2:1 to 7:1 in cell culture mediacomprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS,and 1-5 mM L-glutamine, and added IL-2 at a final concentration of10-200 IU/ml, for a period of 1 to 8 days; (iv) collecting the cellsobtained by step (iii), and; (v) re-stimulating the T cells by culturingcells collected at step (iv) in the presence of EBV-transformed at aresponder to stimulator ratio of 2:1 to 7:1 for a period of 1 to 8 daysin media comprising: (a) cell culture media comprising 40-50% RPMI-1640medium, 40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, (b) atleast 10% conditioned media obtained at the end point of step (iii), and(c) added IL-2 at a final concentration of 10-200 (e.g. 40-100) IU/ml.14. The method according to paragraph 13, wherein the methodadditionally comprises: (vi) collecting the cells obtained by step (v),and; (vii) re-stimulating the T cells by culturing cells collected atstep (vi) in the presence of EBV-transformed LCLs at a responder tostimulator ratio of 2:1 to 7:1 for a period of 1 to 8 days in mediacomprising: (a) cell culture media comprising 40-50% RPMI-1640 medium,40-50% Click's medium, 5-20% FBS, and 1-5 mM L-glutamine, (b) at least10% conditioned media obtained at the end point of step (v), and (c)added IL-2 at a final concentration of 10-200 (e.g. 40-100) IU/ml. 15.The method according to paragraph 13 or 14, wherein the method comprisesadditional steps of collecting cells, and re-stimulating the T cells byculturing the collected cells in the presence of EBV-transformed LCLs(e.g. irradiated, EBV-transformed LCLs) at a responder to stimulatorratio of 2:1 to 7:1 for a period of 1 to 8 days in media comprising: (a)cell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click'smedium, 5-20% FBS, and 1-5 mM L-glutamine, (b) at least 10% conditionedmedia obtained at the end point of the preceding stimulation step, and(c) added IL-2 at a final concentration of 10-200 IU/ml.
 16. A method oftreating a cancer in a subject, the method comprising: (1) isolating Tcells from a subject; (2) generating or expanding a population of Tcells specific for a virus by a method comprising: stimulating T cellsby culture in the presence of antigen presenting cells (APCs) presentinga peptide of the virus, wherein 10 to 25% of the media in which thecells are cultured is conditioned media obtained from a stimulationculture comprising T cells and APCs presenting a peptide of the virus;and (3) administering the generated or expanded population of T cells toa subject.
 17. The method according to claim 16, wherein the conditionedmedia is obtained from a stimulation culture comprising T cells and APCspresenting a peptide of the virus at a responder:stimulator ratio of 2:1to 7:1 for a period of 1 to 8 days.
 18. The method according to claim 16or claim 17, wherein stimulating T cells by culture in the presence ofAPCs presenting a peptide of the virus comprises culture in the presenceof EBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1for a period of 1 to 8 days, in media comprising: (a) cell culture mediacomprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS,and 1-5 mM L-glutamine, (b) 10% to 25% conditioned media obtained by amethod comprising: stimulating T cells by culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 incell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click'smedium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a finalconcentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c)added IL-2 at a final concentration of 10-200 IU/ml.
 19. The methodaccording to claim 16 or claim 17, wherein the APCs presenting a peptideof the virus are EBV-transformed lymphoblastoid cell line (LCL) cells.20. The method according to any one of claims 16 to 19, wherein thecancer is an EBV-positive cancer.
 21. The method according to any one ofclaims 16 to 20, wherein the cancer is EBV-positive nasopharyngealcarcinoma (NPC).
 22. The method according to any one of claims 16 to 21,wherein about 15% of the media in which the cells are cultured isconditioned media.
 23. The method according to any one of claims 16 to22, wherein step (2) additionally comprises: collecting the generated orexpanded population of T cells.
 24. A method of treating a cancer in asubject, the method comprising: (1) isolating T cells from a subject;(2) generating or expanding a population of T cells specific for a virusby a method comprising: stimulating T cells by culture in the presenceof antigen presenting cells (APCs) presenting a peptide of the virus,wherein 10 to 25% of the media in which the cells are cultured isconditioned media, wherein the conditioned media is obtained from astimulation culture comprising T cells and APCs presenting a peptide ofthe virus at a responder:stimulator ratio of 2:1 to 7:1 for a period of1 to 8 days; and (3) administering the generated or expanded populationof T cells to a subject.
 25. The method according to claim 24, whereinstimulating T cells by culture in the presence of APCs presenting apeptide of the virus comprises culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1for a period of 1 to 8 days, in media comprising: (a) cell culture mediacomprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS,and 1-5 mM L-glutamine, (b) 10% to 25% conditioned media obtained by amethod comprising: stimulating T cells by culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 incell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click'smedium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a finalconcentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c)added IL-2 at a final concentration of 10-200 IU/ml.
 26. The methodaccording to claim 24 or claim 25, wherein the cancer is an EBV-positivecancer.
 27. The method according to any one of claims 24 to 26, whereinthe cancer is EBV-positive nasopharyngeal carcinoma (NPC).
 28. Themethod according to any one of claims 24 to 27, wherein the 10% to 25%conditioned media is about 15% conditioned media.
 29. The methodaccording to any one of claims 24 to 28, wherein step (2) additionallycomprises: collecting the generated or expanded population of T cells.30. A method for generating or expanding a population of T cellsspecific for a virus, comprising stimulating T cells by culture in thepresence of antigen presenting cells (APCs) presenting a peptide of thevirus, wherein 10% to 25% of the media in which the cells are culturedis conditioned media obtained from a stimulation culture comprising Tcells and APCs presenting a peptide of the virus.
 31. The methodaccording to claim 30, wherein the conditioned media is obtained from astimulation culture comprising T cells and APCs presenting a peptide ofthe virus at a responder:stimulator ratio of 2:1 to 7:1 for a period of1 to 8 days.
 32. The method according to claim 30 or claim 31, whereinstimulating T cells by culture in the presence of APCs presenting apeptide of the virus comprises culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1for a period of 1 to 8 days, in media comprising: (a) cell culture mediacomprising 40-50% RPMI-1640 medium, 40-50% Click's medium, 5-20% FBS,and 1-5 mM L-glutamine, (b) 10% to 25% conditioned media obtained by amethod comprising: stimulating T cells by culture in the presence ofEBV-transformed LCLs at a responder to stimulator ratio of 2:1 to 7:1 incell culture media comprising 40-50% RPMI-1640 medium, 40-50% Click'smedium, 5-20% FBS, and 1-5 mM L-glutamine, and added IL-2 at a finalconcentration of 10-200 IU/ml, for a period of 1 to 8 days, and (c)added IL-2 at a final concentration of 10-200 IU/ml.
 33. The methodaccording to claim 30 or claim 31, wherein the APCs presenting a peptideof the virus are EBV-transformed lymphoblastoid cell line (LCL) cells.34. The method according to any one of claims 30 to 33, wherein the 10%to 25% of conditioned media is about 15% of conditioned media.
 35. Themethod according to any one of claims 30 to 34, wherein the methodadditionally comprises: collecting the generated or expanded populationof T cells.
 36. The method according to any one of claims 30 to 34,wherein the method additionally comprises: mixing the generated orexpanded population of T cells with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.
 37. A population of T cellsspecific for a virus, wherein the population of T cells is obtained by,obtainable by, or is the product of, a method according to any one ofclaims 1 to 15 or 30 to
 36. 38. A pharmaceutical composition comprisinga population of T cells according to claim 37 and a pharmaceuticallyacceptable carrier, adjuvant, excipient or diluent.
 39. A population ofT cells according to claim 37, or a pharmaceutical composition accordingto claim 38, for use in the treatment or prevention of a disease ordisorder.
 40. Use of a population of T cells according to claim 37, or apharmaceutical composition according to claim 38, in the manufacture ofa medicament or vaccine for use in the treatment or prevention of adisease or disorder.
 41. A method of treating or preventing a disease ordisorder in a subject, comprising administering to a subject atherapeutically or prophylactically effective amount of a population ofT cells according to claim 37, or a pharmaceutical composition accordingto claim
 38. 42. The population of T cells or pharmaceutical compositionfor use according to claim 39, the use according to claim 40, or themethod according to claim 41, wherein the disease or disorder is causedor exacerbated by infection with the virus for which the T cells arespecific, or is a disease or disorder for which infection with the virusfor which the T cells are specific is a risk factor.
 43. The populationof T cells or pharmaceutical composition for use, the use, or the methodaccording to any one of claims 39 to 42, wherein the disease or disorderis a cancer.
 44. The population of T cells or pharmaceutical compositionfor use, the use, or the method according to claim 43, wherein thecancer is an EBV-positive cancer.
 45. The population of T cells orpharmaceutical composition for use, the use, or the method according toclaim 43 or claim 44, wherein the cancer is an EBV-positivenasopharyngeal carcinoma (NPC).
 46. A kit of parts comprising apredetermined quantity of a population of T cells according to claim 37,or the pharmaceutical composition of claim 38.